Development of a multilocus sequence typing method for accurate identification of 16SrV phytoplasma strains via Oxford nanopore

Authors: Costanzo, Stefano; Grinstead, Samuel; ZHAO, YAN - Retired ARS Employee; Wei, Wei

Submitted to: Phytopathogenic Mollicutes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2024
Publication Date: 3/5/2025
Citation: Costanzo, S., Grinstead, S.C., Zhao, Y., Wei, W. 2025. Development of a multilocus sequence typing method for accurate identification of 16SrV phytoplasma strains via Oxford nanopore. Phytopathogenic Mollicutes. 15(1):17-18. https://doi.org/10.5958/2249-4677.2025.00008.2.
DOI: https://doi.org/10.5958/2249-4677.2025.00008.2

Interpretive Summary: This study introduces an innovative method for detecting and identifying strains of elm yellows phytoplasma, a diverse group of bacteria responsible for serious plant diseases across Europe, Asia, and the Americas. Because phytoplasmas cannot be grown outside their hosts plants or insect vectors, their identification relies on molecular techniques and gene sequencing. Most current protocols for identifying phytoplasmas are based on analyzing the sequence of the 16S ribosomal RNA (rRNA) gene. However, the 16S rRNA gene sequence is highly similar across elm yellows phytoplasmas, making it challenging to distinguish between different strains. Researchers from ARS developed a methodology that combines DNA amplification from multiple genes with a portable, real-time DNA sequencing device to analyze infected plant samples. By targeting three specific genes (16S rRNA, secY, and map), they generated enough genetic data in under two hours to accurately identify various elm yellows phytoplasma strains. A key component of this method is the use of molecular barcodes, which enables multiple samples to be processed in a single sequencing run, reducing costs. The study’s findings indicate that this approach can effectively differentiate between closely related strains and even detect mixed infections, with accuracy comparable to traditional methods. This technique could be applied to other harmful plant pathogens and has the potential to become a valuable diagnostic tool for protecting U.S. agriculture and the environment.

Technical Abstract: The elm yellows phytoplasma group (16SrV) includes diverse members associated with devastating plant diseases on a broad host range across Europe, Asia and Americas. To enable the detection and precise identification of a number of 16SrV phytoplasma strains, it was developed and assessed a multilocus sequence analysis approach that combines targeted PCR pre-amplification of DNAs with nanopore sequencing. Three housekeeping genes (16S rRNA; secY; map) were amplified from each sample and sequenced simultaneously using a single-use Flongle Flow Cells. Furthermore, it was developed a bioinformatic analysis pipeline to run BLAST searches of assembled reads against a curated DNA sequence database of 16SrV phytoplasmas. Preliminary results on selected DNA samples suggest that this method can produce sufficient reads and consensus sequence information across the three selected markers to allow accurate determination of phytoplasma strains identity in less than two hours. Moreover, by barcoding the amplicons produced during library preparation, it was possible to process up to 24 different samples in one sequencing experiment therefore reducing the overall cost per sample. Owe to the improved R10.4.1 Flow Cell and the high sequencing depth, consensus sequences produced by this method were at least as accurate as those obtained by Sanger sequencing and allowed differentiation of closely related strains of group 16SrV and the identification of mixed infections.